1. Field of the Invention
The present invention relates to a polypeptide having 25-hydroxyvitamin D.sub.3 -1.alpha.-hydroxylase activity, DNA encoding the polypeptide, a recombinant DNA prepared by inserting the DNA in a vector, a transformant carrying the recombinant DNA, a method for preparing 25-hydroxyvitamin D.sub.3 -1.alpha.-hydroxylase by using the transformant, a method for preparing 1.alpha., 25-dihydroxyvitamin D.sub.3 by using the polypeptide having 25-hydroxyvitamin D.sub.3 -1.alpha.-hydroxylase activity and to an antibody recognizing the polypeptide.
2. Prior Art
Active type vitamin D.sub.3 has been known as a hormone having various biological actions such as the action of controlling calcium metabolism, the induction of cellular differentiation, and immunomodulation.
It has been known that active type vitamin D.sub.3 is generated from vitamin D.sub.3 having no biological actions through the metabolism in biological organisms.
As one of the action mechanisms of active type vitamin D.sub.3 an action mechanism through cytoplasmic receptors have been known.
It has been known that active type vitamin D.sub.3 is essentially 1.alpha., 25-dihydroxyvitamin D.sub.3 wherein the positions 1.alpha. and 25 have been hydroxylated. As to the metabolic pathway for the activation, it has been known that vitamin D.sub.3 is firstly modified into 25-hydroxyvitamin D.sub.3 by introducing a hydroxyl group into the position 25 and the position 1.alpha. of the resulting 25-hydroxyvitamin D.sub.3 is hydroxylated to form 1.alpha., 25-dihydroxyvitamin D.sub.3 [All of vitamin D.sub.3, edited by Etsuro Ogata, Tateo Suda, and Yosuke Ogura, Kodansha Scientific, Co. (1993)].
As 25-hydroxylase gene which functions to introduce a hydroxyl group into the position 25, a gene derived from rat liver has been cloned (Japanese Published Unexamined Patent Application No.2324893/1991). Furthermore, the gene of the hydroxylase of the position 24 of vitamin D.sub.3 has been cloned [Japanese Published Unexamined Patent Application No.207196/1992].
As an enzyme to hydroxylate the position 1.alpha. of vitamin D.sub.3, human CYP27 has been reported [Proc. Natl. Acad. Sci., USA, 91, 10014 (1994)], but the activity of the enzyme to hydroxylate the position 1.alpha. is a secondary activity, so the activity is very weak, which is not an essential activity. Additionally, the activity is not inducible.
It has been known that 25-hydroxyvitamin D.sub.3 -1.alpha.-hydroxylase activity is induced in the kidneys of rats and chickens fed with vitamin D.sub.3 deficient diet [Gerontology, 42 (Supplement 1), 67-77 (1996)].
Up to now, no report has been presented in any of animal species concerning the isolation of any enzyme polypeptide catalyzing the final stage of vitamin D.sub.3 activation to hydroxylate the most significant position 1.alpha., or the isolation of a gene encoding the polypeptide.
As a method for producing 1.alpha.,25-dihydroxyvitamin D.sub.3, a method comprising the use of kidney homogenates or mitochondria fractions of animals such as chicken has been known [Nature, 230, 228 (1971); J. Biol. Chem., 247, 7528 (1972); Biochemistry, 25, 5512 (1986)], but the method requires a vast amount of animal kidney or liver and demands laborious works to prepare them, so the method is insufficient and is not practical. It has been found a microorganism having activity to directly induce hydroxyl groups into the positions 1.alpha. and 25 (Japanese Published Examined Patent Application No.64678/1992), but the activity is very weak and substrate specificity is low, so it is difficult to separate the product and byproducts.